Investigations within this project concern the cell biology of rare human genetic disorders and normal and abnormal intracellular processes. The research goal is to gain insight into changes in molecular function that underlie various genetic metabolic disorders and work towards treatments for these illnesses. The research focuses on three groups of rare disorders: 1. Disorders of sialic acid metabolism. The key enzyme in the sialic acid biosynthesis pathway is UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Dominant mutations in the allosteric site of GNE cause sialuria, characterized by overproduction of sialic acid. Recessive mutations in GNE cause the neuromuscular disorder hereditary inclusion body myopathy (HIBM). In the last year, we wrote an extensive review on all aspects of GNE (Current Biology, under review), identified novel GNE isoforms in human and mice and performed molecular modeling on these isoforms (Biochemistry, under review), performed biochemical analysis for a gene therapy trial of a human HIBM patient (Ref. 5), and defined the kidney phenotype in our HIBM mouse model and used this model to develop a lectin panel for screening human kidney disorders for hyposialylation (American Journal of Pathology, under review). Our further studies focused on subcellular localization and expression levels of GNE and other enzymes in the sialic acid synthesis pathway (Western blotting and real-time quantitative PCR) and identifiying human blood-markers that can serve as parameters for sialylation status (mainly by glycan-profiling studies). In 2007, we characterized a knock-in HIBM mouse model and demonstrated that N-acetylmannosamine (ManNAc) rescues the phenotype of the homozygous mutant mice and is a promising treatment for human patients (J Clin Inv (2007) 117:1585-1594). Negotiations regarding an extensive toxicology study for an IND (investigational New Drug) application for the use of ManNAc are ongoing through the NIH-TRND (Therapies for Rare and Neglected Diseases) program, and our ManNAc patent (No. 60/932,451) is licensed to a ManNAc manufacturer, New Zealand Pharmaceuticals. This last year we further characterized the adult onset muscle phenotype of our HIBM knock-in mouse model and tested alternative HIBM treatments on our murine model, including feeding with sialic acid pathway intermediates and GNE gene therapy, mostly intravenous delivered embedded in liposomes (Lipoplex). The results are being compiled for a manuscript. We described a method of retro-orbital intravenous delivery of compounds to newborn mice (Ref. 3). We filed an employee invention report for the use of liposomes to systemically deliver saccharides (i.e., ManNAc and sialic acid) to mammals. Our mouse model showed an unexpected kidney phenotype (of podocytopathy and glomerular membrane splitting) which was rescued by ManNAc feeding. We developed a lectin panel that characterized the renal glycosylation/sialylation status of our HIBM mouse model (American Journal of Pathology, under review). We are now testing this panel on a variety of unexplained human renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane. Human renal disorders involving glomerular hyposialylation may benefit from ManNAc as a therapeutic agent. 2. Disorders of lysosome-related organelles (LRO) biogenesis. Such disorders include Hermansky-Pudlak syndrome (HPS), Chediak-Higashi syndrome, Griscelli syndrome, Gray Platelet syndrome, and other genetically unclassified disorders. Common clinical features are albinism due to defects in melanosomes and bleeding due to platelet defects. We investigate known and unknown LRO-disorders-causing genes (by conventional and next generation sequencing techniques), with the goal of better understanding the biology of the disease. To study the effects of LRO-disorders mutations, we perform cell biological studies on patient material (using immuno-fluorescence, immmuno-EM, and live cell imaging) to examine defective intracellular trafficking and sorting of proteins and organelles in cells. Such cells fail to transport certain lysosome-related organelle resident proteins to their correct destinations, and LRO-disorder gene products are generally involved in recognizing the specific vesicles that give rise to LROs. We also catalogue the clinical and genetic characteristics of the distinct subtypes of HPS and related LRO-disorders. This last year we identified a patient (second in the world) with HPS subtype 8 (HPS-8);identified a novel human HPS subtype, HPS-9 (Ref. 4);employed SNP-array homozygosity mapping combined with whole exome sequencing to identify disease causing mutations in two different genes in a patient with albinism and neutropenia (Ref. 6);identified by homozygosity mapping and whole exome sequencing NBEAL2 as the long sought-after gene for Gray-Platelet Syndrome (Refs 1 and 7);described new mutations in the HPS1 gene among Puerto-Rican patients (Ref. 2);and described HPS gene mutations in non-Puerto-Rican patients of Hispanic descent (Ref. 8). Furthermore, we characterized Griscelli syndrome type 3 cases (Pigment Cell and Melanoma Research, in press) and describe pulmonary fibrosis in HPS-2 patients (Molecular Medicine, under review). Our group is advises and assists other research groups in the cell biology of metabolic disorders, such as assistance in intracellular localization studies on the ACSF3 gene, found mutated in a subtype of combined malinic and methylmalonic aciduria (Ref. 9). 4. Genetics of Smith-Magenis syndrome (SMS) and related disorders. SMS is a complex neurobehavioral disorder characterized by multiple congenital anomalies, primarily ascribed to a de novo interstitial deletion of 17p11.2. Molecular analysis of SMS patients may shed light on the variable phenotype and genotype-phenotype correlations and possible treatment decisions. The NIH cohort of SMS patients contains patients with the common 3.7 Mb 17p11.2 deletion (n=80), with atypical 17p11.2 deletions (n=24), and non 17p11.2 deleted patients (n= 44). Our whole genome SNP-array analysis on the atypical deletion group identified different 17p11.2 breakpoints that may influence clinical features (manuscript in preparation). SNP-array analysis of the non-deleted cohort identified several novel (micro) deletions or duplications. Our extensive RAI1 gene analysis on the non-deleted subgroup, including mutation analysis and expression studies, identified 10 patients with RAI1 mutations (5 de novo, 5 familial) and described for the first time decreased RAI1 mRNA expression levels not only in patients with the common 17p11.2 deletion but also in RAI1 mutated patients, and in some non-17p11.2 deleted patients (Ref. 10).